1. Field of the Invention
The present invention is related to methods for developing target-specific hybridization sequences, and in particular, to a method for developing hybridization probes using natural nucleotide mismatches and having reduced cross hybridization.
2. Background Art
The ability of DNA microarrays to analyze tens of thousands of genes in one assay has led to continuous improvements in microarray techniques and to software that analyzes microarray data. While researchers have begun to pay close attention to the quality of microarray data, the problem of cross-hybridization has received little consideration.
The success of microarrays in gene expression profiling depends on the specificity between the selected probes and the target genes. All DNA microarrays operate on the principle of DNA hybridization between complementary target and probe sequences. In cross-hybridization, an expressed gene hybridizes with probes designed for other genes as well as with its own designated probe, introducing noise. Experiments with cDNA microarrays have raised cross-hybridization concerns. Although oligonucleotides have the advantage of greater specificity than cDNA microarrays, they too are subject to some degree of cross-hybridization. In particular, genes of highly similar sequences such as those in a large gene family are very difficult to distinguish in microarray experiments.
An even greater problem related to cross-hybridization arises when microarrays are employed to discriminate single nucleotide polymorphism (“SNPs”). In order to enhance specificity, new probes other than conventional linear oligonucleotides have been developed, such as structured DNA probes (molecular beacons) or gold nanoparticle probes. Another approach includes the preparation of target genes by pooling two separate PCRs. Unfortunately, all of these approaches require new materials and/or complex processes. A simpler, more promising approach by Guo et al. involves the introduction of artificial mismatches in the probes to enhance the discrimination of SNPs. However, they introduced artificial nucleotides, which cannot utilize the hybridization differences between natural nucleotides, for mismatch pairs. Moreover, such artificially nucleotides are not appropriate for in vivo applications. Also their dataset was limited in size and only short length oligos were used in the experiments. Moreover, it is difficult to extend their findings to general SNP probe selection.
Accordingly, there is a need in the prior art for systematic methods of designing target-specific hybridization sequences with little or no cross-hybridization.